The first see-through frog created by breeding: description, inheritance patterns, and dermal chromatophore structure.

We have succeeded in creating see-through frogs from natural color mutants of the Japanese brown frog Rana japonica, which usually possesses an ochre or brown back; this coloration enables the organs, blood vessels, and eggs to be observed through the skin without performing dissection. We crossed two kinds of recessive color mutant (black-eyed and gray-eyed) frogs through artificial insemination, and F2 offspring produced frogs whose skin is translucent throughout the life cycle. Three kinds of dermal chromatophores--xanthophores, iridophores, and melanophores--are observed in a layered arrangement in the skin of wild-type frogs, but few chromatophores were present in the skin of the see-through frogs. The translucent skin enables observation of organ growth and cancer formation and progression in the animal, which can be monitored over its entire life without the need for dissection. See-through frogs thus provide a useful animal model for environmental, medical, and biological research.

Hagfish C1q: its unique binding property.

Hagfish C1q (HaC1q) was identified and characterized as a pattern-recognition molecule (PRM) in the hagfish complement system. The serum from hagfish, Eptatretus burgeri, was applied to a GlcNAc-agarose column and eluted sequentially with GlcNAc and EDTA. Four (31, 27, 26, and 19 kDa) and one (26 kDa) proteins were detected as bound molecules in the GlcNAc- and the EDTA-eluates, respectively. Among these, the 26 kDa protein from the EDTA eluate was found to be a homologue of mammalian C1q through cDNA analysis. HaC1q had an ability to bind to various microbes in a Ca(2+)-dependent manner and its target ligands on the microbes were lipopolysaccharide, lipoteichoic acid, and peptidoglycan. The binding of HaC1q to GlcNAc-agarose was not inhibited by an excess amount of monosaccharide such as GlcNAc. While HaC1q bound to Sepharose 6B with a matrix of GlcNAc-agarose (polymer of agarobiose), it did not bind to Sepharose 4B that contained lower concentration of agarobiose than Sepharose 6B. Therefore, the target of HaC1q on GlcNAc-agarose was concluded to be agarobiose and high density of the target moiety seemed to be required for the stable binding. This finding was in accordance with the known behavior of other lectins involved in the complement system. We have concluded that HaC1q recognizes agarobiose-like structures present on the surface of microbes and acts as a pattern-recognition molecule in the process for elimination of invading microbes.

Artificial production and natural breeding of the endangered frog species Odorrana ishikawae, with special reference to fauna conservation in the laboratory.

Odorrana ishikawae is listed as a class IB endangered species in the IUCN Red List and is protected by law in both Okinawa and Kagoshima Prefectures, Japan. Here, in an effort to help effectively preserve the genetic diversity of this endangered species in the laboratory, we tested a farming technique involving the artificial breeding of frogs, and also promoted natural breeding in the laboratory. Field-caught male/female pairs of the Amami and Okinawa Island populations were artificially bred using an artificial insemination method in the 2004, 2006, and 2008 breeding seasons (March to April). Although fewer than 50% of the inseminated eggs achieved metamorphosis, approximately 500, 300, and 250 offspring from the three respective trials are currently being raised in the laboratory. During the 2009 and 2010 breeding seasons, second-generation offspring were produced by the natural mating activities of the first offspring derived from the two artificial matings in 2004. The findings and the methods presented here appear to be applicable to the temporary protection of genetic diversity of local populations in which the number of individuals has decreased or the environmental conditions have worsened to levels that frogs are unable to survive by themselves.

Mitochondrial DNA diversification, molecular phylogeny, and biogeography of the primitive rhacophorid genus Buergeria in East Asia.

In this study we sought to clarify the evolutionary relationships and biogeographic history of the bell-ring frog, Buergeria buergeri (family Rhacophoridae), and two congeneric species Buergeria japonica and Buergeria robusta, by analyzing three mitochondrial (mt) genes: 12S rRNA, Cytb, and ND5. Phylogenetic analyses based on gene data showed the mt clades corresponding to the Buergeria species and three major haplogroups within B. buergeri. Each haplogroup corresponded clearly to the area in which it was distributed, namely eastern Japan (excluding Hokkaido; Hg I), central Japan (Hg II), and western Japan (including the Shikoku and Kyushu regions; Hg III). The estimated divergence time suggested that the lineage splits of the Buergeria species occurred before the formation of the island of Taiwan and the Japan Archipelago (including the Ryukyu islands). The differentiation among the genealogical lineages of B. buergeri seems to have begun in the Late Miocene (approx. 7-5Mya), and the formation of their present distribution pattern might have been influenced by climatic changes and geographical events such as the formation of a wide peneplane and expansions of certain basins.

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae.

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus.

Complete mitochondrial genomes and novel gene rearrangements in two dicroglossid frogs, Hoplobatrachus tigerinus and Euphlyctis hexadactylus, from Bangladesh.

We determined the complete nucleotide sequences of mitochondrial (mt) genomes from two dicroglossid frogs, Hoplobatrachus tigerinus (Indian Bullfrog) and Euphlyctis hexadactylus (Indian Green frog). The genome sizes are 20462 bp in H. tigerinus and 20280 bp in E. hexadactylus. Although both genomes encode the typical 37 mt genes, the following unique features are observed: 1) the ND5 genes are duplicated in H. tigerinus that have completely identical sequences, whereas duplicated ND5 genes in E. hexadactylus possessed dissimilar substitutions; 2) duplicated control region (CR) in H. tigerinus has almost identical sequences whereas single control region (CR) was found in E. hexadactylus; 3) the tRNA-Leu (CUN) gene is translocated from the LTPF tRNA cluster to downstream of ND5-1 in H. tigerinus, and the tRNA-Pro gene is translocated from the LTPF tRNA cluster to downstream of CR in E. hexadactylus; 4) pseudo tRNA-Leu (CUN) and tRNA-Pro genes are observed in E. hexadactylus; and 5) two tRNA-Met genes are encoded in both species, as observed in the previously reported dicroglossid mt genomes. Almost all observed gene rearrangements in H. tigerinus and E. hexadactylus can be explained by the tandem duplication and random loss model, except translocation of tRNA-Pro in E. hexadactylus. The novel mt genomic features found in this study may be useful for future phylogenetic studies in the dicroglossid taxa. However, the mt genome with interesting features found in the present study reveal a high level of variation of gene order and gene content, inspiring more research to understand the mechanisms behind gene and genome evolution in the dicroglossid and as well as in the amphibian taxa in future studies.

[An analysis of fall accidents using Kochi Fire Department ambulance statistics].

PURPOSE: The aim of this study was to examine ambulance usage due to fall accidents in Kochi City. METHOD: The survey period was one year, from January to December, 2005. A total of 967 ambulance responses to fall accidents were recorded by the Kochi Fire Department during this period. Ambulance responses to fall accidents were analyzed in terms of sex, age, time, and place of fall. Adults were defined as subjects between the ages of 18 and 64; elderly adults were defined as being 65 or older. RESULTS: The number of ambulance responses per a population sample of 1000 people was 1.32 cases in adults (men 1.55, women 1.11) and 10.48 cases in elderly adults (men 9.14, women 11.32). The proportion of outdoor falls for adults was 60.8%, for women and 56.8% for men. The respective values for elderly adults were 54.9% and 36.6%. The majority of elderly adult women fell down indoors. More falls occurred in winter (from December to February) than in the other seasons. There were more falls in December than other months. CONCLUSION: Fall incidence varies according to sex, age and season. We can conclude that the causes and places of falls are related to physical activity and season. Fire department ambulance records can be utilized to analyze fall situation in local areas. Such analyses should lead to new approaches to fall prevention measures.

Complete nucleotide sequence of the mitochondrial genome of Schlegel's tree frog Rhacophorus schlegelii (family Rhacophoridae): duplicated control regions and gene rearrangements.

The complete nucleotide sequence (21,359 bp) of the mitochondrial DNA of the rhacophorid frog Rhacophorus schlegelii was determined. The gene content, nucleotide composition, and codon usage of this genome corresponded to those typical of vertebrates. However, the Rh. schlegelii genome was unusually large due to the inclusion of two control regions and the accumulation of lengthy repetitive sequences in these regions. The two control regions had 97% sequence similarity over 1,510 bp, suggesting the occurrence of concerted sequence evolution. Comparison of the gene organizations among anuran species revealed that the mitochondrial gene arrangement of Rh. schlegelii diverged from that of typical vertebrates but was similar to that of Buergeria buergeri. The positions of the tRNA-Leu(CUN) and tRNA-Thr genes were exchanged between Rh. schlegelii and B. buergeri. Based on parsimonious consideration and the basal phylogenetic position of B. buergeri, these genes seemed to have been rearranged in an ancestral lineage leading to Rh. schlegelii.

Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the bell-ring frog, Buergeria buergeri (family Rhacophoridae).

In this study we determined the complete nucleotide sequence (19,959 bp) of the mitochondrial DNA of the rhacophorid frog Buergeria buergeri. The gene content, nucleotide composition, and codon usage of B. buergeri conformed to those of typical vertebrate patterns. However, due to an accumulation of lengthy repetitive sequences in the D-loop region, this species possesses the largest mitochondrial genome among all the vertebrates examined so far. Comparison of the gene organizations among amphibian species (Rana, Xenopus, salamanders and caecilians) revealed that the positioning of four tRNA genes and the ND5 gene in the mtDNA of B. buergeri diverged from the common vertebrate gene arrangement shared by Xenopus, salamanders and caecilians. The unique positions of the tRNA genes in B. buergeri are shared by ranid frogs, indicating that the rearrangements of the tRNA genes occurred in a common ancestral lineage of ranids and rhacophorids. On the other hand, the novel position of the ND5 gene seems to have arisen in a lineage leading to rhacophorids (and other closely related taxa) after ranid divergence. Phylogenetic analysis based on nucleotide sequence data of all mitochondrial genes also supported the gene rearrangement pathway.